A Selective, Slow Binding Inhibitor of Factor VIIa Binds to a Nonstandard Active Site Conformation and Attenuates Thrombus Formation in Vivo

Olivero, Alan G.; Eigenbrot, Charles; Goldsmith, Richard; Robarge, Kirk; Artis, Dean R.; Flygare, John A.; Rawson, Thomas E.; Sutherlin, Daniel P.; Kadkhodayan, Saloumeh; Beresini, Maureen H.; Elliott, Linda O.; DeGuzman, Geralyn G.; Banner, David W.; Ultsch, Mark; Marzec, Ulla M.; Hanson, Stephen R.; Refino, Canio J.; Bunting, Stuart; Kirchhofer, Daniel

doi:10.1074/jbc.m409068200

Cited by 40 publications

(46 citation statements)

References 72 publications

(38 reference statements)

Supporting

Mentioning

Contrasting

Order By: Relevance

“…This is overcome either by the fusion of the two peptides (51) or by using a protease switch with substrate phage (49). A number of synthetic compounds have also been designed as active-site inhibitors of FVIIa as well as the TF⅐FVIIa complex (52,(55)(56)(57)(58). A number of naphthylamidines have recently been reported to have FVIIa inhibitory activity (59).…”

Section: Discussionmentioning

confidence: 99%

Hemextin AB Complex, a Unique Anticoagulant Protein Complex from Hemachatus haemachatus (African Ringhals Cobra) Venom That Inhibits Clot Initiation and Factor VIIa Activity

Banerjee

Mizuguchi

Iwanaga

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

During injury or trauma, blood coagulation is initiated by the interaction of factor VIIa (FVIIa) in the blood with freshly exposed tissue factor (TF) to form the TF⅐FVIIa complex. However, unwanted clot formation can lead to death and debilitation due to vascular occlusion, and hence, anticoagulants are important for the treatment of thromboembolic disorders. Here, we report the isolation and characterization of two synergistically acting anticoagulant proteins, hemextins A and B, from the venom of Hemachatus haemachatus (African Ringhals cobra). N-terminal sequences and CD spectra of the native proteins indicate that these proteins belong to the three-finger toxin family. Hemextin A (but not hemextin B) exhibits mild anticoagulant activity. However, hemextin B forms a complex (hemextin AB complex) with hemextin A and synergistically enhances its anticoagulant potency. Prothrombin time assay showed that these two proteins form a 1:1 complex. Complex formation was supported by size-exclusion chromatography. Using a "dissection approach," we determined that hemextin A and the hemextin AB complex prolong clotting by inhibiting TF⅐FVIIa activity. The site of anticoagulant effects was supported by their inhibitory effect on the reconstituted TF⅐FVIIa complex. Furthermore, we demonstrated their specificity of inhibition by studying their effects on 12 serine proteases; the hemextin AB complex potently inhibited the amidolytic activity of FVIIa in the presence and absence of soluble TF. Kinetic studies showed that the hemextin AB complex is a noncompetitive inhibitor of soluble TF⅐FVIIa amidolytic activity, with a K i of 50 nM. Isothermal titration calorimetric studies showed that the hemextin AB complex binds directly to FVIIa with a binding constant of 1.62 ؋ 10 5 M ؊1 . The hemextin AB complex is the first reported natural inhibitor of FVIIa that does not require a scaffold to mediate its inhibitory activity. Molecular interactions of the hemextin AB complex with FVIIa/TF⅐FVIIa will provide a new paradigm in the search for anticoagulants that inhibit the initiation of blood coagulation.

show abstract

Section: Discussionmentioning

confidence: 99%

Hemextin AB Complex, a Unique Anticoagulant Protein Complex from Hemachatus haemachatus (African Ringhals Cobra) Venom That Inhibits Clot Initiation and Factor VIIa Activity

Banerjee

Mizuguchi

Iwanaga

et al. 2005

Journal of Biological Chemistry

View full text Add to dashboard Cite

show abstract

“…The K i values were calculated according to the relationship K i ϭ IC 50 /(1 ϩ [S]/K m ) (23) using the experimentally determined IC 50 and K m values for each enzyme-substrate pair. For HAI-2, the apparent K i values (K i *) were determined by fitting the data to the equation for tight binding inhibition (24,25), 2 , 0.05 mg/ml BSA, 5 mM glucose), and 0.8 ml of 100 nM pro-uPA in prewarmed HBSA-glucose buffer was added to the cell layers. The inhibitors KD1 or HI-10331 were added to give final concentrations of 1 and 10 M, respectively.…”

Section: Real-time Reverse Transcription-pcr-totalmentioning

confidence: 99%

Pro-urokinase-type Plasminogen Activator Is a Substrate for Hepsin

Moran¹,

Li²,

Fan³

et al. 2006

Journal of Biological Chemistry

Self Cite

View full text Add to dashboard Cite

Hepsin, a type II transmembrane serine protease, is strongly up-regulated in prostate cancer. Hepsin overexpression in a mouse prostate cancer model resulted in tumor progression and metastasis, associated with basement membrane disorganization. We investigated whether hepsin enzymatic activity was linked to the basement membrane defects by examining its ability to initiate the plasminogen/plasmin proteolytic pathway. Because plasminogen is not processed by hepsin, we investigated the upstream activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator. Enzymatic assays with a recombinant soluble form of hepsin demonstrated that hepsin did not cleave pro-tissue-type plasminogen activator but efficiently converted pro-uPA into high molecular weight uPA by cleavage at the Lys 158 -Ile 159 (P 1 -P 1 ) peptide bond. uPA generated by hepsin displayed enzymatic activity toward small synthetic and macromolecular substrates indistinguishable from uPA produced by plasmin. The catalytic efficiency of pro-uPA activation by hepsin (k cat /K m 4.8 ؋ 10 5 M ؊1 s ؊1 ) was similar to that of plasmin, which is considered the most potent pro-uPA activator and was about 6-fold higher than that of matriptase. Conversion of pro-uPA was also demonstrated with cell surface-expressed full-length hepsin. A stable hepsinoverexpressing LnCaP cell line converted pro-uPA into high molecular weight uPA at a rate of 6.6 ؎ 1.9 nM uPA h ؊1 , which was about 3-fold higher than LnCaP cells expressing lower hepsin levels on their surface. In conclusion, the ability of hepsin to efficiently activate pro-uPA suggests that it may initiate plasmin-mediated proteolytic pathways at the tumor/stroma interface that lead to basement membrane disruption and tumor progression.Hepsin is a type II transmembrane serine protease expressed on the surface of epithelial cells. The 417-amino acid protein is composed of a short N-terminal cytoplasmic domain, a transmembrane domain, and a single scavenger receptor cysteinerich domain that packs tightly against the C-terminal protease domain (1). The physiologic function of hepsin remains unknown. Despite its expression in the very early stages of embryogenesis (2), hepsin-deficient mice were viable and developed normally (3, 4). The studies further showed that hepsin was not essential for liver regeneration and for coagulationrelated physiological functions (3, 4). However, hepsin has been implicated in ovarian cancer (5) and prostate cancer (6 -11), where several gene expression studies have identified it as one of the most highly induced genes. Hepsin RNA levels were found to be low in normal prostate and benign hyperplasia but strongly increased in prostate carcinoma, particularly in advanced stages (8 -10). Hepsin protein staining with a monoclonal anti-hepsin antibody showed that hepsin expression was highest at sites of bone metastasis and in late stage primary tumors (12), which is consistent with the finding that increased hepsin RNA levels correlated with higher Gleason grades...

show abstract

“…14,15 Interaction with the catalytic Ser195 via addition of a -amino group on the benzamidine contributes significantly to potency in a number of inhibitors. 8,16 In contrast, a series of compounds was shown to form a unique network of hydrogen bonds to Ser195 via a hydroxyl group on a non-P1 central ring. 17,18 Another P1 substitution reported is the addition of an o-OH on -aminobenzamidine to increase selectivity.…”

Section: State-of-the Artmentioning

confidence: 99%

“…Therefore there is a critical unmet medical need for improved oral anticoagulants with a better therapeutic window. Direct comparisons of specific FVIIa/TF inhibitors with other anticoagulants in preclinical thrombosis models have shown a reduced bleeding tendency in several studies, [3][4][5][6][7][8] suggesting that targeting the upstream proteolytic complex FVIIa/TF may provide a safety advantage. Such a drug would need to be potent, orally bioavailable, and highly selective for FVIIa/TF versus other closely related serine proteases involved in coagulation and fibrinolysis, as well as demonstrate other optimal pharmacokinetic parameters.…”

mentioning

confidence: 99%

Inhibitors of Factor VIIa/Tissue Factor

Shirk

Vlasuk

2007

ATVB

View full text Add to dashboard Cite

Abstract-The formation of the proteolytic complex composed of the serine protease Factor VIIa and the cell-associated glycoprotein tissue factor (FVIIa/TF) initiates a cascade of amplified zymogen activation reactions leading to thrombus formation. The critical role of the coagulation cascade in pathological thrombosis has been the basis for significant efforts to design selective inhibitors of the protease components as new anticoagulant alternatives for the treatment of thrombotic diseases. However, for the new generation of anticoagulant drugs in development that primarily target protease complexes distal from FVIIa/TF, the differential between efficacy and safety as defined by bleeding is unresolved. Targeting the FVIIa/TF complex has several theoretical advantages that exploit the amplified nature of the coagulation cascade. However, progress on the development of clinical-stage FVIIa/TF-based anticoagulants has not been as successful to date. This review summarizes recent efforts in the discovery of synthetic inhibitors of FVIIa/TF. Key Words: anticoagulants Ⅲ factor VIIa (FVIIa) Ⅲ tissue factor Ⅲ serine protease inhibitors Ⅲ coagulation H emostasis is achieved through a highly regulated and coordinated interplay of protein and cellular components designed to respond quickly to vascular insult. Critical in this rapid response is the initiation of blood coagulation by a proteolytic complex composed of the serine protease factor VIIa (FVIIa) and its cell-associated cofactor tissue factor (TF). Tissue factor is a transmembrane glycoprotein that is normally found in subendothelial structures throughout the vasculature. Exposure of TF to blood, through physical damage to the lining of the blood vessel or increased expression in endothelial and monocytic cells in response to certain inflammatory signals, permits an interaction with FVIIa, which is present at trace levels in the circulation. The formation of the FVIIa/TF complex is the critical step that initiates the sequentially amplified cascade of proteolytic activation steps that ultimately leads to the generation of the protease thrombin and thrombus formation. 1 The critical nature of the FVIIa/TF complex in the process of blood coagulation makes it an attractive candidate for the development of antithrombotic agents that can inhibit complex formation or catalytic activity. An antithrombotic agent based on the inhibition of FVIIa/TF may have theoretical advantages over the inhibition of downstream coagulation proteases such as factor Xa (FXa) or thrombin based on the several thousand-fold amplification that occurs after initiation by FVIIa/TF. The success of several small protein-and antibody-based inhibitors of the FVIIa/TF pathway in preclinical models and clinical trials 1,2 has led to substantial interest in the development of orally active small molecule inhibitors of the enzyme active site that could be used for the long-term prevention or treatment of thrombosis. Vitamin K antagonists such as warfarin, which have been used clinically for more than 5...

show abstract

A Selective, Slow Binding Inhibitor of Factor VIIa Binds to a Nonstandard Active Site Conformation and Attenuates Thrombus Formation in Vivo

Cited by 40 publications

References 72 publications

Hemextin AB Complex, a Unique Anticoagulant Protein Complex from Hemachatus haemachatus (African Ringhals Cobra) Venom That Inhibits Clot Initiation and Factor VIIa Activity

Hemextin AB Complex, a Unique Anticoagulant Protein Complex from Hemachatus haemachatus (African Ringhals Cobra) Venom That Inhibits Clot Initiation and Factor VIIa Activity

Pro-urokinase-type Plasminogen Activator Is a Substrate for Hepsin

Inhibitors of Factor VIIa/Tissue Factor

Contact Info

Product

Resources

About